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More than 100,000 Detectable Peptide Species Elute in Single Shotgun Proteomics Runs but the Majority is Inaccessible to Data-Dependent LC−MS/MS

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Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Proteome Res. 2011, 10, 4, 1785-1793
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    More than 100,000 Detectable Peptide Species Elute in Single Shotgun Proteomics Runs but the Majority is Inaccessible to Data-Dependent LC−MS/MS
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    Department for Proteomics and Signal Transduction at the Max-Planck Institute of Biochemistry, Martinsried, Germany
    *Matthias Mann, Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18 D-82152, Martinsried, Germany. E-mail: [email protected]. Fax: +49 89 8578 2219.
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    Journal of Proteome Research

    Cite this: J. Proteome Res. 2011, 10, 4, 1785–1793
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    https://doi.org/10.1021/pr101060v
    Published February 10, 2011
    Copyright © 2011 American Chemical Society
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    Abstract

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    Shotgun proteomics entails the identification of as many peptides as possible from complex mixtures. Here we investigate how many peptides are detectable by high resolution MS in standard LC runs of cell lysate and how many of them are accessible to data-dependent MS/MS. Isotope clusters were determined by MaxQuant and stringently filtered for charge states and retention times typical of peptides. This resulted in more than 100 000 likely peptide features, of which only about 16% had been targeted for MS/MS. Three instrumental attributes determine the proportion of additional peptides that can be identified: sequencing speed, sensitivity, and precursor ion isolation. In our data, an MS/MS scan rate of 25/s would be necessary to target all peptide features, but this drops to less than 17/s for reasonably abundant peptides. Sensitivity is a greater challenge, with many peptide features requiring long MS/MS injection times (>250 ms). The greatest limitation, however, is the generally low proportion of the target peptide ion intensity in the MS/MS selection window (the “precursor ion fraction” or PIF). Median PIF is only 0.14, making the peptides difficult to identify by standard MS/MS methods. Our results aid in developing strategies to further increase coverage in shotgun proteomics.

    Copyright © 2011 American Chemical Society

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    Cite this: J. Proteome Res. 2011, 10, 4, 1785–1793
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